This Program Project proposes in-depth studies of the development of CD8+ T lymphocyte responses to persistent (EBV) and non-persistent (vaccinia, yellow fever) virus infections with particular emphasis on understanding induction and long-term maintenance of antigen-specific and cross-reactive CD8+ T cells. The overall goals are: 1) To use models of persistent (EBV) and non-persistent (VV or YFV) viral infections to characterize the lineage relationship between effector and memory responses in humans and the factors that influence evolution of antigen-specific CD8+ T cell responses into the memory CD8+ T cell repertoire;2) To characterize how cross-reactivity influences the evolution of the antigen-specific CD8+ T cell repertoire and understand the structural and functional properties of the T cell receptor that contribute to recognition of antigen-specific or cross-reactive ligands;3) To correlate antigen-specific and cross-reactive CD8+ T cell frequencies and functional properties with control of viral replication or manifestations of disease. Project #1 will use the Epstein Barr model of persistent viral infection to characterize the lineage relationship between effector and memory responses and the factors that influence evolution of antigen-specific CD8+ T cell responses into the memory CD8+ T cell repertoire. Project #2 will test the hypothesis that the quantity, quality, and avidity of an epitope-specific response to acute viral infection are dramatically altered by acute heterologous virus infections and may alter disease outcome. It will also test the hypothesis that, because of prior antigenic exposure, adults may have higher frequencies of cross-reactive CD8+ T cells than children. TCR alpha and beta chain spectratyping and sequencing will allow improved understanding of the structural bases for the recognition of antigen-specific or cross-reactive ligands. Project #3 will study vaccinia virus or yellow fever virus vaccine recipients to characterize the lineage relationship between effector and memory responses in humans and the factors that influence evolution of antigen-specific CD8+ T cell responses into the memory CD8+ T cell repertoire. This project will also test the hypothesis that VV or YFV infection will reactivate cross-reactive CD8+ T cells. The proposed work will benefit from a highly interactive and collaborative group of productive investigators. Four core facilities will facilitate the work of Project Investigators. The Administrative and Clinical Core (Core A) will coordinate the Projects;manage fiscal affairs;be responsible for the collection and processing of clinical specimens from research subjects with AIM or in the latent phase of EBV infection;provide EBV serology and molecular HLA Class I and II typing of research subjects. The Tetramer Core (Core B) will provide HLA Class l/peptide tetramers to all Project Investigators. The Flow Cytometry (Core C) will provide FACS-based cell sorting and characterization of virus-specific or cross-reactive CD8+ T cell responses. A TCR Core (Core D) will perform spectratyping, TCR subcloning and sequencing, maintain a TCR repertoire database, and facilitate TCR data analyses. The proposed studies will help us to understand the timing of and factors that control the evolution of human antiviral CD8+ T cell responses from the acute effector phase into long-term memory. These studies will also help us to understand variability in human responses to viral infections and how antigen-specific or cross-reactive CD8+ T cells contribute to either viral control or disease. Understanding the mechanisms operative in the evolution of virus-specific CD8+ T cell responses should contribute to the development of new or improved viral vaccines and the development of novel therapies for autoimmune diseases or virus associated malignancies. PROJECT 1: "Evolution and Maintenance of EBV-Specific Memory CD8+ T Cells" (Luzuriaga, K.) PROJECT 1 DESCRIPTION (provided by applicant): Work outlined in this proposal addresses the hypotheses that virus-specific CD8+ T cell repertoires change quantitatively and qualitatively over time following acute infection, and that differences in virus-specific CD8+ T cell repertoires may influence the control of viral replication or disease pathogenesis. Changes in the EBV-specific CD8+ T cell repertoire over time may be based on properties of EBV-specific CD8+ T cell clones (specificity, T cell receptor usage, cytokine receptors, and expression of survival factors) and ongoing antigen exposure. Collaborative work with Drs. Selin and Welsh (Project 3) will test the hypothesis that age-related differences in the evolution of cross-reactive CD8+ T lymphocyte responses will be observed and may be influenced by prior (or lack of prior) exposure to other viral pathogens. To address these hypotheses, we will investigate the evolution of CD8+ T lymphocyte responses during the early (induction) and long-term (maintenance) phases of the immune response in infants, children, adolescents, and adults. The following Specific Aims will be addressed: 1) To define the relationship between the virus-specific T cell receptor repertoires at serial time points from the acute through the memory phases of the T cell response;2) To define the relationship between the expression of cytokine receptors or survival factors during the acute phase of the T cell response and T cell clonal persistence into the memory pool;3) To define the relationship between the functional profiles of virus-specific T cells during the acute phase of the T cell response and their maintenance of proliferative and cytolytic capacity during the long-term memory phase;and 4) To correlate EBV-specific CD8+ T cell frequencies and functional properties over time with blood viral load, pharyngeal shedding, and disease severity. This project directly supports the overall objective of the Program Project by characterizing the induction, evolution, and maintenance of virus-specific CD8+ T lymphocyte responses to a persistent viral infection. Our investigation of memory CD8+ T lymphocyte responses resulting from a persistent viral infection with repeated but changing exposure to viral antigens will complement Drs. Rothman's and Ennis'investigation of memory CD8+ T lymphocyte responses resulting from receipt of yellow fever or vaccinia vaccines in Project 3. Altogether, these studies will help us to understand the timing of and factors that control the evolution of human antiviral CD8+ T cell responses from the acute effector phase into long-term memory. These studies will also help us to understand how antigen-specific or cross-reactive CD8+ T cells may contribute to either viral control or disease. Ultimately, we hope that they will help to inform the development of antiviral vaccines that afford long-term protection against infection or disease, while minimizing the potential adverse effects of these vaccines.